This invention relates to new hydridomas (hybrid cell lines), namely ATCC HB 8418 and ATCC HB 8426, filed Nov. 9, 1983 and Nov. 16, 1983, respectively. More specifically, this invention concerns the production of monoclonal antibodies from each of such new hybridomas such antibodies specific to in vivo fragments derived from fibrinogen, and to diagnostic and therapeutic methods and compositions employing these antibodies.
Fibrinogen is a large (M.sub.r 340,000) dimeric molecule composed of three non-identical polypeptide chains. The fibrinogen-fibrin transition involves the sequential release of fibrinopeptides. In this two-stage thrombin-mediated process, fibrin I is the initial product and it is formed following the release of FPA [fibrinopeptide A (A.alpha. 1-16)]. Fibrin II, a more compact structure, results upon the release of FPB [fibrinopeptide B (B.beta. 1-14)] (Blomback et al., Nature, Lond., 257, 501-505, 1978). Dissolution of fibrin--be it fibrin I or II--deposits is required in order to restore vascular integrity. This is achieved principally via the plasmin pathway (see Kernoff and McNicol, Br. Med. Bull., 33, 239-244, 1977 and Collen, Thromb. Haemostos., 43, 77-89, 1980). One of the early plasmin degradation products of fibrinogen or fibrin is derived from the NH.sub.2 -terminal portion of the B.beta. chain. The bond B.beta. 42 Arg-43 Ala is particularly susceptible to plasmin (Mosesson et al., J. Biol. Chem., 247, 5210-5219, 1972; Takagi and Doolittle, Biochemistry, 14, 940-946, 1975). The nature of the B.beta. chain peptide released by plasmin depends upon the available substrate. Cleavage of fibrinogen or fibrin I will result in the FPB containing peptide B.beta. 1-42. The latter, of course, cannot be generated in plasmin proteolysis of fibrin II, which results in the release of peptide B.beta. 15-42.
Identification and quantitation of thrombin and plasmin degradation products of fibrinogen and fibrin may serve as valuable diagnostic tests. During the past decade, a number of immunoassays have been developed for this purpose. Antisera prepared by conventional immunization have been used in most of these assays. Since such antisera contain antibodies of varying titer, affinity and specificity, a number of problems have been encountered. For example, antibodies to cleavage-associated neo-antigens found on certain fragments of fibrinogen and fibrin have generally been present in extremely low titer (Plow and Edgington, J. Clin. Invest., 52, 273-282, 1983; J. Biol. Chem., 250, 3386-3392, 1975). Regarding cross-reactivity, most antisera react with intact fibrinogen and, therefore, plasma samples require a processing step(s) in order to selectively remove it. Significant differences in immunoreactivity have been observed for antisera prepared to both FPA and FPB (Canfield et al., Biochemistry, 15, 1203-1208, 1976; Wilner et al., Biochemistry, 15, 1209-1213, 1976; Bilezikian et al., J. Clin. Invest., 56, 438-445, 1975). Some of these antisera show limited cross-reactivity with peptides that are only a few amino acid residues longer than free FPA or FPB.
The development of the hybridoma technique by Kohler and Milstein, Nature, 256, 495-497, 1975, may allow refinement of most of the immunoassays dealing with fibrinogen or fibrin degradation products.
The fusion of mouse myeloma cells to spleen cells from immunized mice by Kohler and Milstein demonstrated for the first time that it was possible to obtain a continuous cell line making monoclonal antibody. Subsequently, much effort has been directed to the production of various hybrid cells (hybridomas) and to the use of the antibody made by these hybridomas. See, for example, F. Melchers, M. Potter, and N. Warner, eds., Current Topics in Microbiology and Immunology, 81-"Lymphocyte Hybridomas", Springer-Verlag, 1978, and the references contained therein; C. J. Barnstable, et al., Cell, 14, 9-20, May, 1978; P. Parham and W. F. Bodmer, Nature, 276, 397-399 November, 1978; D. M. Wier, ed., Handbook of Experimental Immunology, Third Edition, 2, Blackwell, 1978, Chapter 25; and Chemical and Engineering News., Jan. 1, 1979, 15-17. These references indicate the problems inherent in attempting to produce monoclonal antibodies from hybridomas. While the general technique is well understood, there are many difficulties and variations in each specific case.
In fact, there is no assurance, prior to attempting to prepare a given hybridoma, that the desired hybridoma will be obtained, that it will produce antibody if obtained, or that the antibody so produced will have the desired specificity. The degree of success is influenced principally by the type of antigen employed and the selection technique used for isolating the desired hybridoma.
Prior research had shown that cleavage of fibrinogen in vitro with CNBr results in the release of a major NH.sub.2 -terminal fragment, the so-called N-DSK (Blomback et al., Nature, 218, 130-134, 1968). The N-DSK portion of fibrinogen contains binding or polymerization domains which are exposed as a consequence of enzyme activation and which are operative in the fibrinogen to fibrin transition (Kudryk et al., J. Biol. Chem., 249, 3322-3325, 1974). The enzyme thrombin can cleave FPA an FPB from fibrinogen, and release of both peptides results in the formation of fibrin II. In contrast to thrombin, the snake venom enzyme Bathroxobin can release only FPA from fibrinogen (Laurent & Blomback, Acta Chem. Scand., 12, 1875-1877, 1958), a process which leads to the formation of a type of fibrin which has been termed fibrin I. Since the NH.sub.2 -terminal ends of fibrinogen and N-DSK are identical, different N-DSK species can be obtained from thrombin and Batroxobin induced fibrin gels.
In hope of identifying neoepitopes, Qureshi et al., Thromb. Res., 6, 357-374, 1975, prepared rabbit antisera to human (T)N-DSK. Despite the fact that high titer sera were obtained, these investigators could not demonstrate any immunochemical differences between N-DSK and (T)N-DSK.
In 1982, Kudryk et al., developed a radioimmunoassay which could be used to measure the plasma levels of peptides containing the B.beta. 15-42 sequence derived from fibrinogen or fibrin (Kudryk et al., Thromb. Res., 25, 277-291, 1982). Since these peptides arise as a consequence of in vivo plasmin digestion, it was proposed that the assay could give important information in clinical studies on disease states where thrombosis was imminent or manifest. However, the assay could not distinguish between peptides containing extensions at either the NH.sub.2 - or COOH-terminal end of B.beta. 15-42 and, therefore, only the total B.beta. 15-42 immunoreactivity could be measured.
Nossel et al., J. Clin. Invest., 64, 1371-1378, 1979, have identified B.beta. 1-42 in clinical blood samples and suggested that it arises from plasmin proteolysis of the so-called fibrin I. A second type of fibrin is also formed in vivo. By definition, fibrin II lacks both FPA and FPB and, therefore, its dissolution by plasmin cannot generate B.beta. 1-42 (See FIG. 8 herein). Nossel (Nature, Lond., 291, 165-167, 1981) has suggested that fibrin I is the pivotal substrate in fibrinogen proteolysis in vivo and that fibrin II is more likely to result in occlusive thrombosis (see FIG. 8 herein). Since occlusive thrombosis imperils a patient's life, the incipiency of thrombosis must be detected. The degradation of fibrinogen in vivo results in products which cause thrombosis, depending on the prevalent enzymatic reactions occurring during fibrinogen proteolysis. Problems have existed in that it has been difficult to determine in any reliable manner, whether the fibrin I polymer which results from thrombin activation of fibrinogen is, in turn, activated by thrombin or plasmin. If activated primarily by plasmin, split products, including a fragment of the B.beta. chain of fibrin I containing amino acid residues 1-42, are produced. If the latter is the predominant pathway, occlusive thrombosis does not occur. If fibrin I polymer is, instead, further activated by thrombin, fibrin II is formed and this is often accompanied by thrombosis.
In light of the above it has become very desirable to determine which biochemical route the fibrin I molecule is taking. One approach for doing this is to identify the molecular nature of the B.beta. chain peptides in clinical samples. For example, confirmation of the predominance of intact B.beta. 1-42 in a patient's blood plasma, with a coupled negative indication of peptide fragments of the B.beta. chain containing amino acid residues 1-14 and 15-42, would strongly suggest plasmin protolysis of fibrin I. On the other hand, the reverse finding would indicate that fibrin I had been further degraded to yield fibrin II. As mentioned above, plasmin degradation of fibrin II can never yield B.beta. 1-42.
Matsuedo et al., Science, 222, 1129-1132, 1983, developed three monoclonal antibodies that bind to human fibrin from hybridomas prepared from fusion of cells of a Sp 2/O myeloma cell line and spleen cells from a female BALB/c mouse immunized with a complex consisting of a synthetic heptapeptide of the amino terminus of the B.beta. chain of human fibrin, a cysteine residue placed at the heptapeptide's carboxy terminus, and MB-KLH (maleimidobenzoylated keyhole limpet hemocyanin). One of the monoclonal antibodies cross-reacted with fibrin from species other than human, e.g., rabbit fibrin.